Irf3R278Q/R278Qmice exhibit elevated viral load in the CNS. (A) Schematic of the ocular infection route and establishment of HSE-like disease in mice. Image was created in BioRender. Virus replicates in the trigeminal ganglion with spreading to the brain stem. (B–D) Eyes (B), TG (C), and brain stems (D) were harvested on the indicated time points from WT (n = 5–7) and Irf3R278Q/R278Q (n = 5–12) mice infected with HSV-1 McKrea (2 × 106 PFU/eye). Viral titer determined by plaque assay on tissue homogenates. (E) Viral titer in brain stem homogenates isolated on day 5 from WT (n = 8), Irf3WT/R278Q (n = 8), and Irf3R278Q/R278Q (n = 11) mice infected with HSV-1 McKrea (2 × 106 PFU/eye). (F) Representative confocal microscopy images of HSV-1 foci in the brain stems of WT and Irf3R278Q/R278Q on day 5 after infection (blue: DAPI, red: HSV-1 ICP5, white: CD45, and green: cleaved-caspase 3). Magnification, 10×; scale bar, 50 μm. (G–I) Quantification of images from Fig. 2 E of number of (G) HSV-1 ICP5+, (H) CD45+, and (I) cleaved caspase-3+ cells per image (UI, n = 16; WT, n = 24; Irf3R278Q/R278Q, n = 20 images, respectively) Brain stems imaged: UI, n = 4; WT, n = 6; Irf3R278Q/R278Q, n = 5. Each brain stem is represented by images of two different anatomic locations (−5.80 mm and −6.34 mm, relative to bregma, respectively), two images per brain stem: left and right side lesions, respectively. Scale bar, 50 μm. (J–L) Levels of HSV-1 gB transcripts 24 h after infection (MOI 1.0) in vitro in murine (J) microglia, (K) astrocytes, and (L) neurons were assessed by RT-qPCR of HSV-1 gB normalized to β-actin (Actb). (M and N) HSV-1 titers in supernatants harvested from human iPSC-derived (M) astrocytes and (N) cortical neurons 24 h after infection with MOI 0.01–1, assessed by TCID50% assay. Viral replication kinetics (B–D) were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype). Error bars; SEM. Statistical analyses of virus titer, image quantification, and CNS cell experiments (E, G–I, and J–N) were performed by two-tailed one-way ANOVA, followed by unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001; N.D., not detected; CL-Casp3, cleaved caspase-3.
Figure 2.

Irf3 R278Q/R278Q mice exhibit elevated viral load in the CNS. (A) Schematic of the ocular infection route and establishment of HSE-like disease in mice. Image was created in BioRender. Virus replicates in the trigeminal ganglion with spreading to the brain stem. (B–D) Eyes (B), TG (C), and brain stems (D) were harvested on the indicated time points from WT (n = 5–7) and Irf3R278Q/R278Q (n = 5–12) mice infected with HSV-1 McKrea (2 × 106 PFU/eye). Viral titer determined by plaque assay on tissue homogenates. (E) Viral titer in brain stem homogenates isolated on day 5 from WT (n = 8), Irf3WT/R278Q (n = 8), and Irf3R278Q/R278Q (n = 11) mice infected with HSV-1 McKrea (2 × 106 PFU/eye). (F) Representative confocal microscopy images of HSV-1 foci in the brain stems of WT and Irf3R278Q/R278Q on day 5 after infection (blue: DAPI, red: HSV-1 ICP5, white: CD45, and green: cleaved-caspase 3). Magnification, 10×; scale bar, 50 μm. (G–I) Quantification of images from Fig. 2 E of number of (G) HSV-1 ICP5+, (H) CD45+, and (I) cleaved caspase-3+ cells per image (UI, n = 16; WT, n = 24; Irf3R278Q/R278Q, n = 20 images, respectively) Brain stems imaged: UI, n = 4; WT, n = 6; Irf3R278Q/R278Q, n = 5. Each brain stem is represented by images of two different anatomic locations (−5.80 mm and −6.34 mm, relative to bregma, respectively), two images per brain stem: left and right side lesions, respectively. Scale bar, 50 μm. (J–L) Levels of HSV-1 gB transcripts 24 h after infection (MOI 1.0) in vitro in murine (J) microglia, (K) astrocytes, and (L) neurons were assessed by RT-qPCR of HSV-1 gB normalized to β-actin (Actb). (M and N) HSV-1 titers in supernatants harvested from human iPSC-derived (M) astrocytes and (N) cortical neurons 24 h after infection with MOI 0.01–1, assessed by TCID50% assay. Viral replication kinetics (B–D) were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype). Error bars; SEM. Statistical analyses of virus titer, image quantification, and CNS cell experiments (E, G–I, and J–N) were performed by two-tailed one-way ANOVA, followed by unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001; N.D., not detected; CL-Casp3, cleaved caspase-3.

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